Regulators of apoptosis

Apoptotic signaling is strictly regulated, ensuring a balance between repair and destruction, cell life and death. Many factors are involved in this process.

The most important ones will be shortly introduced in the following part.

III.3.1. IAPs

Eight human inhibitors of apoptosis proteins (IAPs) proteins have been identified which share homology to the originally identified baculovirus IAPS.

The main function of IAPs is the inhibition of activated caspases (Fig.1) through direct interaction of the baculovirus IAP repeat (BIR) domain within the IAPs to active caspases (Takahashi et al., 1998; Salvesen, 2002a;).

Different BIR domain can bind to different caspases. In case of XIAP, the BIR3 domain binds directly to the small subunit of caspase-9, whereas the BIR2 domain binds to caspase-3 and -7 (Huang et al., 2001; Srinivasula et al., 2001). IAP function is controlled by SMAC/Diablo. SMAC/Diablo contains a tetrapeptide motif with which it binds to IAPs and frees bound caspases (Srinivasula et al., 2001; Verhagen et al., 2000).

III.3.2. Bcl-2 family members

B cell lymphoma-2, the founding member of the Bcl-2 family, was first identified in chromosomal breakpoint of t(14;18) bearing human follicular B cell lymphoma (Bakhshi et al., 1985; Cleary and Sklar, 1985; Tsujimoto et al., 1985). The members of the Bcl-2 family can be divided in three groups according to their structural homology to the four Bcl-2 homology domains (BH1-4) and function (Figure 3).

The antiapoptotic members contain all four homology domains and include BCL-2, BCL-X^L (Boise et al., 1993), MCL-1 (Desagher et al., 1999; Wei et al., 2001) A1 (Choi et al., 1995), and BCL-W (Gibson et al., 1996). The first pro-apoptotic Bcl-2 family member, the Bcl-2 associated X protein (BAX) was identified by its binding to Bcl-2 (Oltvai et al.,1993) and forms together with BAK and BOK the multi-domain pro-apoptotic Bcl-2 family which contain the homology domains BH1-BH3. The localization of Bax and Bak differs,

while Bax is mainly cytosolic or loosely associated with the mitochondria, Bak is an integral part of the ER- and the mitochondrial membrane, bound to VDAC (Adams and Cory., 2007). The third Bcl-2 family subgroup contains the BH3-only members Bim, Bad, Bik, Bik, Bmf, HRK, Noxa, Puma. All members of this group are pro-apoptotic and share the signature BH-3 domain.

Figure 3: Bcl-2 family proteins and their subdivision. (From Adams and Cory, 2001)

Apoptosis induction via the intrinsic pathway is mainly regulated by Bcl-2 family members. After stress-induction, Bax and Bak are thought to travel to the mitochondria and by oligomerization and forming of a mitochondrial apoptosis-induced channel (MAC), perforate the outer mitochondrial membrane (Liu et al., 1996; Pavlov et al.,2001; Wei et al., 2001; De Giorgi et al., 2002; Guo et al., 2004). Bax and Bak are in most tissues largely redundant. Thus the absence of one protein has no effect on cell death, but the absence of both proteins inhibits apoptosis in many cell types (Wei et al, 2001, Dejean et al, 2005). Pro-survival Bcl-2-family proteins bind Bax and Bak, thereby inhibiting their translocation to the mitochondria and MAC formation (Pavlov et al., 2001; Guo et al., 2004; Martinez-Caballero et al.,2004). The BH3-only proteins act upstream of Bax and Bak, as they

2001; Zong et al., 2001). Two models which describe the apoptosis induction by BH-3 only proteins have been proposed.

The direct activation model (Figure 4) suggests, that the activators tBid and Bim bind directly to Bax and Bak and therby activate them (Letai et al., 2002;

Kuwana et al., 2005; Certo et al., 2006; Oh et al, 2006; Walensky et al., 2006). The other BH3-only members, the sensitizers, free tBid and Bim from the Bcl-2 family members, enabling the activation of Bax and Bak. Several findings make the direct activation model unlikely. No binding of Bak to any of the BH3-only members could be found and even in the absence of tBid and Bim apoptosis was not impaired (Willis et al., 2007).

The indirect activation model (Figure 4) proposes that the BH3-only family members bind to the pro-survival Bcl-2 proteins and thereby release Bax and Bak (Chen et al., 2005; Willis et al., 2005; Willis et al, 2007). The importance of single BH3-only family members depends on their ability to bind pro-survival proteins. Bim and tBid are able to bind all pro-pro-survival Bcl-2 family members and are therefore more potent than the other BH3-only proteins which can only bind to a subset of the pro-survival proteins.

The concentration and the activity of BH3-only proteins changes under stress conditions, whereas the levels of Bax and Bak are unaltered. The activation of p53 induces the expression of Noxa and Puma (Jeffers et al., 2003;

Villunger et al., 2003; Erlacher et al., 2005). Bim is induced downstream of the Akt pathway and its activity is regulated by phosphorylation (Dijkers et al., 2000). Phophorylation by Erk triggers Bim degradation, whereas phosphorylation by c-Jun N-terminal kinase potentiates its pro-apoptotic activity (Ley et al., 2003; Luciano et al., 2003; Akiyama et al., 2003; Putcha et al., 2003). The BH-3 only protein Bid needs to be proteolytically processed by caspases or granzyme B.

Figure 4: Contrasting direct and indirect activation models for Bax and Bak.

(a) In the direct model (Letai et al., 2002), the putative activators Bim and tBid bind directly to Bax and Bak to drive their activation, whereas the sensitizers only bind to the pro-survival Bcl-2 homologs (‘Bcl-2 et al.’) via the BH3 domain (red triangle). (b) In the indirect activation model (Chen et al., 2005; Willis et al., 2005, 2007), the BH3-only proteins activate Bax and Bak not by binding to them directly, but instead by engaging the multiple pro-survival proteins that guard Bax and Bak. In this model, Bim and tBid are more potent than Bad and other BH3-only proteins owing to the greater range of pro-survival proteins that they can engage and neutralize (from Adams and Cory, 2007).